ELISA enzyme-linked immunosorbent assay

ELISA (enzyme-linked immunosorbent assay) is a technique that has long been applied to the detection of clinical protein markers in body fluid samples and is therefore used as a "gold standard", but is in fact subject to a simple experimental paradigm and limited optimization measures. The sensitivity of ELISA technology is very limited. The ELISA also shows different detection efficiencies due to the different antibodies used. Crude polyclonal antibodies, affinity purified polyclonal antibodies, and monoclonal antibodies are widely used in ELISA. It is often necessary to test the specificity, sensitivity, etc. of the antibody when constructing the kit. Western Blot testing of the same antibody using different samples can help us determine the specificity of the antibody. Comparing and optimizing antibodies from various sources plays a fundamental role in the development of ELISA kits.

Multiplex Multiple Marker Detection Technology

ELISA technology is very mature and easy to grasp, but its sensitivity and low throughput are attracting researchers' attention and trying to develop better techniques to replace this traditional method. The multi-factor or multi-analyte simultaneous identification technique is of great significance to researchers because of the large need to obtain cheaper data, or when the sample size is limited and more data is needed.

Representative multiplex biomarker detection techniques include planar matrix detection and liquid suspension chips. The planar matrix detection is represented by the solid phase protein chip of R&D and other companies. By immunocapturing different standards or samples at different positions of the solid phase chip, the spatial position is used to realize the identification of different samples, and the enzyme-labeled secondary antibody is produced. The color reaction response is reported by the specific analyte content at each sample point, and the instrument can judge the content of different analytes by simple card reading. The low weight of the solid phase protein chip analysis and the narrow dynamic range make the application of this technology limited, which is more suitable for qualitative detection or relative content judgment.

The liquid suspension chip is represented by the Luminex platform, and the CBA technology of companies such as BD can also be classified as such. The Luminex liquid suspension chip uses a red-infrared two-color/trichrome dye mixed fluorescence code to achieve recognition of approximately 100/500 different identity microspheres. When a particular color of microspheres is coupled to an antibody that detects a particular indicator, the different microspheres can work independently in the same sample to capture different markers. The secondary antibody used in this technique is still marker-specific with a biotin tag and can be combined with a streptavidin-phycoerythrin chromogenic reagent. Therefore, the key to the development of this technology is to have antibody pairs that target different epitopes of the same antigen molecule. When the results are tested, the different microspheres incubated with the same sample are illuminated by two independently working lasers. The red laser is used to identify the identity of the microsphere, telling the instrument which indicator is being detected. Another green laser ignites the phycoerythrin, thereby relying on the fluorescence yield to determine the analyte content. CBA and Luminex have a similar encoding method, the difference is that the same laser is used to judge the identity of the microsphere and determine the analyte content. In the aspect of fluorescence compensation and instrument adjustment, higher requirements are placed on the operator. Subject to physical characteristics, the number of analytes detected by the kit is also relatively small, typically around 10 weights. In contrast, thanks to the synergistic work of the two lasers, Luminex can reduce physical interference to a lower degree, and theoretically achieve simultaneous detection of 500 weights. The bottleneck in developing multiple detection kits is no longer an optical property, but a cross-reactivity of different antibodies. Although various attempts have been made to minimize the cross-reactivity of different antibody pairs, in actual kit development work, if the high data quality requirements are adhered to, the highest acceptable detection weight is about 40 about. When a liquid suspension chip is applied to nucleic acid detection, since the cross-reaction caused by the antibody is not considered, the detection number can be greatly increased.

The liquid suspension chip has obvious and powerful value in the actual inspection work. First of all, the liquid suspension chip has a larger detection range (actually between 3.0 and 4.5) while achieving close sensitivity to ELISA, and has better detection efficiency. Secondly, up to 40 different indicators can be achieved in as low as 25 microliters of sample, which can resolve the contradiction between the amount of sample and the amount of data encountered in a large number of studies. Again, lower detection prices and less labor input increase the level of popularity of the technology. Finally, the detection of different indicators in the same sample can be a good reference for the internal reference of different indicators in the sample, and obtain data with unique value. The shortcoming of the liquid suspension chip is that there is no fundamental breakthrough in sensitivity relative to ELISA, and the mutual interference of different reactions in multi-indicator detection brings potential data risk. Therefore, for projects with high data requirements, especially in the field of medical research such as drug research and development, the weakness of liquid suspension chips can not be ignored.

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